RNA-Seq Work Flows

Martin Morgan, Sonali Arora
October 28, 2014

7-step work flow

1. Experimental design

Keep it simple

Replicate

Avoid confounding experimental factors with other factors

Record co-variates

Be aware of batch effects

2. Wet-lab

Confounding factors

Artifacts of your particular protocols

3. Sequencing

Axes of variation

Application-specific, e.g.,

4. Alignment

Alignment strategies

Splice-aware aligners (and Bioconductor wrappers)

(5a. Bowtie2 / tophat / Cufflinks / Cuffdiff)

5. Reduction to 'count tables'

Step 6. Analysis

Summarization

Normalization

Appropriate error model

Pre-filtering

Borrowing information

Step 7. Comprehension

Placing differentially expressed regions in context

Lab

The lab is based on a modified version of the RNA-seq work flow developed by Michael Love, Simon Anders, Wolfgang Huber.